Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found within and at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Atherosclerosis is a leading cause of death in the United States and results from the formation of plaques in arterial walls that often occlude the vessel lumen and obstruct blood flow. Morbidity and mortality generally occur through end organ damage and organ dysfunction resulting from ischemia. The most common forms of ischemic end organ damage are myocardial infarction and cerebrovascular accidents. Disability or death often result from these vascular events. Atherosclerosis-related ischemia that does not permanently injure myocardium often causes anginapectoris and congestive heart failure. Additionally, atherosclerotic occlusions may damage other organs, such as the kidneys, the intestines, and the spinal cord. These occlusions consist of plaques that form by accumulation of cholesterol, cholesterol esters, and phospholipids and the proliferation of smooth muscle cells in the intima of major arteries. Lipid contributes a major portion of the plaque volume (generally 30-65% dry weight). In fact, the risk of developing arteriosclerosis is directly related to the concentration of certain forms of plasma cholesterol. The reduction of the initiation and progression of atherosclerosis by eliminating accumulation of lipid within vessel walls provides a therapeutically valuable approach for the prevention of cardiovascular disease.
Delivery of cholesterol into cells occurs via the receptor-mediated low density lipoprotein (LDL) pathway and by passive exchange of sterol between plasma membranes and lipoproteins. Only tissues that produce steroid hormones and bile acids can metabolize cholesterol. In order to prevent accumulation of excess free sterol in remaining peripheral tissues there is a reverse transport of cholesterol from plasma membranes into HDL and lipoprotein-like particles. HDL transports excess cholesterol to the liver where it can either be processed into bile salts for excretion or incorporated into very low density lipoproteins (VLDL) to re-enter the lipoprotein pool.
Assays that are currently available to measure cholesterol movement often employ isotopically labeled cholesterol to measure the movement of unesterified cholesterol (free cholesterol, FC) into (influx) or out of (efflux) cells. However, these assays have several disadvantages. First, the movement of cholesterol between cells and serum is bidirectional, thus net cholesterol flux is determined by the relative contribution of cholesterol influx and cholesterol efflux. Currently available assays measure cholesterol efflux only, and therefore fail to account for cholesterol influx. Measurements obtained from these assays could be inaccurate and misleading. Second, because these currently available assays measure the efflux of unesterified cholesterol, they cannot account for the pool of EC in atherogenic cells which is the major form of cholesterol present in foam cells both in vitro and in vivo (3, 4). Finally, inasmuch as non-human macrophage cells are used in such assays, they are not ideally suited for testing therapeutic agents for the treatment of human subjects.